Assignment of the [4Fe-4S] clusters of Ech hydrogenase from Methanosarcina barkeri to individual subunits via the characterization of site-directed mutants
Assignment of the [4Fe-4S] clusters of Ech hydrogenase from Methanosarcina barkeri to individual subunits via the characterization of site-directed mutants
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DOI：
10.1111/j.1742-4658.2005.04889.x
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发表时间：
2005-09-01
期刊：
影响因子：
5.4
通讯作者：
Hedderich, R
中科院分区：
文献类型：
--
作者：
Forzi, L;Koch, J;Hedderich, R
来自巴氏甲烷八叠球菌（Methanosarcina barkeri）的Ech氢化酶是一组不同的膜结合[NiFe]氢化酶的成员，其序列与节能的NADH：醌氧化还原酶（复合物I）相似。该酶的序列预测三个[4Fe-4S]簇的结合，一个由亚基EchC和两个由亚基EchF。先前的研究表明，这些簇中的两个可以在pH 7下在10（5）Pa的H-2下完全还原，产生两个不同的S1/2电子顺磁共振（EPR）信号，指定为g = 1.89和g = 1.92信号。在不同pH值下的氧化还原滴定表明，这两个集群有一个pH依赖的中点电位指示离子泵的功能。为了将这些信号分配给酶的亚基，一组M。产生了巴氏突变体，其中EchF中8个保守的半胱氨酸残基中的7个被丝氨酸单独取代。从分离的突变体酶记录的EPR谱显示了g = 1.92信号的强烈减少或完全丧失，而g = 1.89信号在5种突变体酶中仍然可检测到作为主要EPR信号。得出的结论是，集群引起的g = 1.89的信号是位于EchC的近端集群和g = 1.92的信号结果从亚基EchF的集群之一。这两个[4Fe-4S]团簇的pH依赖性表明它们同时介导电子和质子转移，因此可能是质子转移机制的重要组成部分。
Ech hydrogenase from Methanosarcina barkeri is a member of a distinct group of membrane-bound [NiFe] hydrogenases with sequence similarity to energy-conserving NADH:quinone oxidoreductase (complex I). The sequence of the enzyme predicts the binding of three [4Fe-4S] clusters, one by subunit EchC and two by subunit EchF. Previous studies had shown that two of these clusters could be fully reduced under 10(5) Pa of H-2 at pH 7 giving rise to two distinct S1/2 electron paramagnetic resonance (EPR) signals, designated as the g = 1.89 and the g = 1.92 signal. Redox titrations at different pH values demonstrated that these two clusters had a pH-dependent midpoint potential indicating a function in ion pumping. To assign these signals to the subunits of the enzyme a set of M. barkeri mutants was generated in which seven of eight conserved cysteine residues in EchF were individually replaced by serine. EPR spectra recorded from the isolated mutant enzymes revealed a strong reduction or complete loss of the g = 1.92 signal whereas the g = 1.89 signal was still detectable as the major EPR signal in five mutant enzymes. It is concluded that the cluster giving rise to the g = 1.89 signal is the proximal cluster located in EchC and that the g = 1.92 signal results from one of the clusters of subunit EchF. The pH-dependence of these two [4Fe-4S] clusters suggests that they simultaneously mediate electron and proton transfer and thus could be an essential part of the proton-translocating machinery.